Large scale purification of castanospermine

ABSTRACT

The invention concerns a scalable, large-scale castanospermine preparation method that yields levels of purity greater than 98% from castanospermine-containing plant sources, such as Castanospermum australe seed material.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 63/034,747, filed Jun. 4, 2020, which is herebyincorporated by reference herein in its entirety, including any figures,tables, nucleic acid sequences, amino acid sequences, or drawings.

BACKGROUND OF THE INVENTION

Castanospermine is an alkaloid produced by the Castanospermum australetree and is enriched in its seeds. Castanospermine is analpha-glucosidase inhibitor which inhibits glycoprotein processing inmammalian cells by preventing removal of 3-glucose residues linked to ahigh mannose core structure. This Glc3Man9GlcNAc2 structure istransferred enblock to an N-linked site in a glycoprotein (N-X-S/T,where N is glutamine, X is any amino acid except Proline, followed byeither a Serine or Threonine). After transfer, alpha-glucosidase removesthe three glucose residues and allowing further processing by multiplemannosidases to reach a core capable of conversion to the complex typeoligosaccharide structure catalyzed by multiple glycosyltransferasesthat is found on most glycoproteins. By inhibiting the initial step,castanospermine prevents glycoprotein processing to yield thecomplex-type structure. This processing occurs in the endoplasmicreticulum and is very often required in order for proper protein foldingto occur and yield a physiologically active protein.

This activity of castanospermine has an antiviral effect on many virusesgiven that many viruses have coat glycoproteins and it has been shownthat in many instances misfolded proteins lacking activity occur withcastanospermine treatment. Thus, castanospermine is a potentialanti-viral therapeutic (Whitby K et al., J Virol., July 2005,79(14):8698-8706; U.S. Pat. No. 10,561,642).

An analytical, small-scale method for producing castanospermine is known(Hohenschutz L et al., 1981, Phytochemistry, 20:811-814). To purifylarge quantities of castanospermine needed for large-scale therapeuticuse, a preparation method tailored to the needs of large-scaleproduction, would be advantageous.

BRIEF SUMMARY OF THE INVENTION

The method of the invention is a scalable, large-scale castanosperminepreparation method. The preparation method differs in significant waysfrom methods for small-scale analytical isolation of castanospermine andis capable of yielding levels of purity greater than 98%.

One aspect of the invention is a method for preparing castanospermine,comprising:

(a) separating a liquid fraction comprising castanospermine from solidplant material, such as Castanospermum australe seed material;

(b) purifying the castanospermine in the liquid fraction from (a) by:(i) mixing the liquid fraction with an ion exchange medium (e.g., ionexchange resin or ion exchange polymer) under conditions that allowcastanospermine in the liquid fraction to be bound to the resin, or (ii)column chromatography using a chromatography medium;

(c) eluting the castanospermine from the ion exchange medium of (b)(i)or chromatography medium of (b)(ii) to produce an eluate; and

(d) purifying the castanospermine in the eluate of (c) by mixing theeluate with an ion exchange medium (e.g., ion exchange resin or ionexchange polymer) under conditions that allow castanospermine in theeluate to be bound to the ion exchange medium, and eluting thecastanospermine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chromatogram from typical conditions for LC/MS analysisof castanospermine (see Example). The purity of castanospermine can beassessed based upon the chromatogram.

FIG. 2 : shows mass spectra (MH+: 190.1, MH−H2O+: 172.2, MH−2H2O+:154.2, MH−3H2O+: 136.1, M+Na+: 212.1) produced under typical conditions(see Example). The quantity of castanospermine can be assessed basedupon the size of MH+ ion at 190.1 AMU in comparison with dilutions and astandard curve.

DETAILED DESCRIPTION OF THE INVENTION

The invention concerns a method for preparing castanospermine,comprising:

(a) separating a liquid fraction comprising castanospermine from solidplant material, such as Castanospermum australe seed material;

(b) purifying the castanospermine in the liquid fraction from (a) by:(i) mixing the liquid fraction with an ion exchange medium (e.g., ionexchange resin or ion exchange polymer) under conditions that allowcastanospermine in the liquid fraction to be bound to the resin, or (ii)column chromatography using a chromatography medium;

(c) eluting the castanospermine from the ion exchange medium of (b)(i)or chromatography medium of (b)(ii) to produce an eluate; and

(d) purifying the castanospermine in the eluate of (c) by mixing theeluate with an ion exchange medium (e.g., ion exchange resin or ionexchange polymer) under conditions that allow castanospermine in theeluate to be bound to the ion exchange medium, and eluting thecastanospermine.

Preparation of Raw Seed Material

The plant material can be prepared by cutting, breaking up, and/orgrinding, producing ground particles, preferably of a size no largerthan about 2 millimeters in diameter. Thus, the plant material may be“processed” into pieces or particulate form. In some embodiments, theplant material is homogenized. Any homogenization method may be usedthat is appropriate for the scale of the material being processed.

Any castanospermine-containing plant material may potentially beutilized as source material for the preparation method, from one or morecastanospermine-containing plants, and from one or morecastanospermine-containing parts of the plant(s), such as seeds, stem,leaf, pod, etc. If seeds having seed cases are used, the seed cases aretypically removed, leaving just the seed, as described in the Example.

Examples of castanospermine-containing plants that may be used includeCastanospermum australe, as was used in the Example, and Alexa spp.Examples of castanospermine-containing Alexa species include A.canaracunensis Pittier, A. cowanii Yakovlev, A. grandiflora Ducke, A.herminiana Ramirez, A. imperatricis (R.H. Schomb.) Baill., A. leiopetalaSandwith, A. superba RS Cowan, and A. wachenheimii Benoist.

In some embodiments, the plant material comprises one or more parts ofC. australe, such as C. australe seeds or leaf. In some embodiments, theplant material comprises C. australe seeds lacking seed cases.

Further details concerning preparation of the solid material areprovided in the “Grinding seeds” section of the Example. It should beunderstood that, although the Example refers to C. australe seeds, anyother castanospermine-containing plant species and plant parts maypotentially be substituted.

Separation of Liquid Fraction

Once the plant material is processed (i.e., broken up, cut, and/orground into pieces particulate matter, and preferably homogenized), themethod comprises separating a liquid fraction containingcastanospermine. The separating step (a) includes an extraction, such asan ethanol extraction, to obtain a liquid fraction containingcastanospermine, and solid material. Various ethanol concentrations maybe used for the ethanol extraction, provided the final ethanolconcentration is sufficient to precipitate the starch from the plantmaterial. For example, the final ethanol concentration may be at leastabout 60%, depending upon the amount of water in the plant material(e.g., the amount of water in seeds). In some embodiments, the finalethanol concentration is in the range of 70% to 75%. In someembodiments, the separating step (a) comprises: (i) allowing the solidmaterial to settle out and placing (e.g., pumping) most of the liquid(first recovered fraction) into a container (e.g., a vat or othervessel) followed by filtration or centrifugation of the remainingmaterial to remove the rest of the liquid fraction (second recoveredfraction) and combining the second recovered fraction with the firstrecovered fraction; or (ii) directly separating the liquid fraction fromthe solid material using a continuous process such as centrifugation orfiltration.

Typically, the solid material recovered from the first extraction willbe a moist cake. Optionally, the separating step (a) is repeated on thesolid material one or more times, pooling the liquid fractions obtainedfrom each separation. Depending upon the specific separation method usedto obtain the liquid fraction, there may be very fine particlesremaining in the liquid fraction. This will settle out and can beremoved from the liquid prior to purifying step (b), or at a later step.Further details concerning preparation for the separation step areprovided in “1. Grinding seeds” and “2. Homogenizing ground seed meal”sections of the Example.

Optional Alternative Heat Treatment

This optional heat treatment has the advantage of not using ethanol forextraction from the plant material (e.g., seeds), and would just beutilized for the extraction phase.

The procedure involves heating raw castanospermine-containing plantmaterial (e.g., Castanospermum seeds) to an internal temperaturesufficient to denature starch and other components (e.g., about 63 toabout 77 degrees C.). Preferably, the plant material is then to cool,e.g., to room temperature or ambient temperature. This heat treatmentwill render the starch and other components mostly insoluble in water(by contrast, when ethanol is used in extraction fluid, it is largelythere to precipitate starch and other components so they can be filteredaway more easily). Castanospermine is stable to this heat treatment.

The cooled plant material (e.g., seeds) can then be ground or cut up toyield pieces and/or particles about 2 to 4 mm in diameter and placed inwater (e.g., deionized water). This mixture can either be stirred tokeep the seed pieces and/or particles suspended or more aggressivelymixed to break the pieces up, for example, in a blender. The more gentlestirring will require more extraction time for the castanospermine toleach out into the water, for example, overnight but will leave solidresidue that will settle out on its own and not require filtration orcentrifugation to obtain a clear or slightly turbid aqueous solutioncontaining extracted castanospermine. More aggressive mixing or blendingwill not require an extensive extraction time for the castanospermine tobecome dissolved in water but, after allowing solid residue to settle,the aqueous solution may have considerable particulate material tofilter or centrifuge out. The solid residue can optionally bere-extracted with additional deionized water.

From either option the pooled aqueous solution can then be mixed with anion exchange medium (such as a Dowex 50 H+ form ion exchange resin orother strongly acidic cation exchange resin), either batch-wise withstirring, or poured into a chromatography column, as described below.After mixing with the ion exchange medium or passing through thechromatography column with chromatography medium, the aqueous solutionis discarded and the resin extensively washed with water (e.g.,deionized water) and eluted with 2N NH₄OH as described elsewhere.Subsequent steps are the same as if ethanol had been used for theinitial extraction.

Purifying Castanospermine in Liquid Fraction

The initial purifying step (b) comprises purifying the castanosperminein the liquid fraction from (a) by: (i) mixing the liquid fraction withan ion exchange medium (e.g., ion exchange polymer or ion exchangeresin, such as DOWEX-50 H⁺ form ion exchange resin or other stronglyacidic cation exchange resin) under conditions that allowcastanospermine in the liquid fraction to be bound to the resin, or (ii)column chromatography using a chromatography medium. In someembodiments, the ion exchange medium of step (b) comprises ion exchangebeads.

Strongly acidic mediums such as DOWEX-50 H⁺ contain exchangeable H+cations which give the resins the ability to behave as insoluble, butvery reactive acids. Not only can these resins be used to exchange theH+ ion for the cations of a salt in solution, effectively “saltsplitting” and forming the acid of the salt involved in solution, butthey can be used in place of soluble acid in many catalytic reactions.DOWEX cation resins include standard gel products such as DOWEX HCR-S,DOWEX HCRW2, DOWEX HGR and DOWEX HGR-W2, macroporous products such asDOWEX M-31, uniform particle size products such as DOWEX G-26, and driedcatalysts such as DOWEX DR 2030.

Optionally, the method includes sampling the mixture after mixing for aduration of time in (b)(i), and measuring the amount of castanospermineremaining in the liquid phase (such as by liquid chromatography massspectrometry (LC/MS)).

Further details concerning the initial purifying step of (b) areprovided in “3. Treatment with Dowex-50H⁺ form” section of the Example.

Elution of Castanospermine

In step (c), the elution of castanospermine from the ion exchange mediumof (b)(i) or chromatography medium of (b)(ii) in the initialpurification step of (b) is carried out in an elution vessel, such as acolumn, to produce an eluate (one or more castanospermine-containingeluted fractions). Ammonium hydroxide (NH₄OH) or other suitable elutionsolvent may be used. As the elution solvent moves through the elutionvessel, the heat of neutralization will create warming, which allows theprogress of the elution to be easily monitored. For example, heatsensitive tape affixed to the side of the vessel may be used. Thecastanospermine elutes from vessel in the region of the warming frontand fractions can be collected of any practical size. Alternatively, itis possible to carry out the elution step of (c) in a batchwise fashion,and separating the castanospermine-containing supernatant fraction fromthe ion exchange medium or chromatography medium using any suitablemethod.

Optionally, the method includes assaying the amount of castanosperminein the eluted fractions (e.g., by LC/MS) and pooling those fractionswith desired high amounts of castanospermine present in order tominimize the amount of impurities carried to the next step.

Optionally, the method further comprises removing residual solvent afterthe elution of (c) and before the purifying step of (d). Because it ispossible that some elution solvent (e.g., NH₄OH) could interfere withthe efficient castanospermine binding in the subsequent purificationstep of (d), residual solvent can be removed by any suitable methodcapable of removing the residual solvent, such as drying orconcentration. For example, a rotary evaporator may be used forconcentration of solvent.

Optionally, the ion exchange medium may be regenerated back to the H+form, and can be used one or more times for absorption ofcastanospermine from additional liquid extracts of seed material fromstep (a) following the same procedures described above.

Further details concerning the elution step of (c) are provided in the“4. Elution of castanospermine from Dowex 50 H⁺ form” section and in the“5. Preparation of the castanospermine containing Dowex 50 H+ formeluted fractions for subsequent steps” section of the Example.

Purifying Castanospermine in Eluate

The purification step of (d) comprises purifying the castanospermine inthe eluate of (c) by mixing the eluate with an ion exchange medium(e.g., ion exchange resin or ion exchange polymer) under conditions thatallow castanospermine in the eluate to be bound to the ion exchangemedium, and eluting the castanospermine. This step can be carried outbatch-wise, or over a column or other vessel. Preferably, the ionexchange medium of the purifying step of (d) comprises a pyridinium ionform resin, wherein castanospermine is exchanged with the pyridiniumion, resulting in castanospermine bound to the resin and pyridinereleased into solution. The castanospermine may be eluted in thepurifying step (d) using an elution solvent, such as pyridine indeionized water, or NH₄OH.

The purity of castanospermine that may be achieved using the purifyingstep of (d) can be least about 95% if the castanospermine is eluted by acolumn or other vessel, or about 90% to about 95% if the castanospermineis eluted batch-wise.

Further details concerning the purification step of (d) are provided inthe “6. Purification on Dowex 50 pyridinium ion form resin” section ofthe Example.

Optionally, the purification step of (d) further comprises furtherpurifying the eluate. The step can include: (i) repeating the purifyingstep of (d) on the eluate, or (ii) recrystallizing the castanospermineenriched crystals in the eluate of step (d) with a solvent, such asethanol, optionally sonicating, placing the solution at reducedtemperature to stimulate crystal formation, for harvesting and drying.

Further details concerning the further purification step of (d) areprovided in the “7. Further purification of castanospermine” section ofthe Example.

Optionally, the ion exchange medium of step (d) can be regenerated oneor more times for repeated use. Details concerning the regeneration stepare provided in the “8. Regeneration of Dowex 50 resin used incastanospermine purification” section of the Example.

The method of the invention may further comprise detecting the presenceof castanospermine, or measuring the amount of castanospermine (e.g., toassess purity), in the product of one or more of steps (a), (b), (c), or(d). In some embodiments, liquid chromatography mass spectrometry(LC/MS) is utilized. Further details concerning LC/MS are provided inthe “9. LC/MS assay for castanospermine” section of the Example.

As is well-known in the art, the steps in a cycle of chromatography candiffer depending on the chromatography resin, the buffers used toperform each step in the cycle, and the biophysical characteristics of atarget compound, such as castanospermine. For example, an affinitychromatography column can include the steps of loading an affinitychromatography column with a fluid including castanospermine, washingthe column to remove unwanted material, eluting the castanosperminebound to the column, and re-equilibrating the column. A cycle ofchromatography, using a cationic and/or anionic exchange chromatographycolumn, where the castanospermine binds to the chromatography resin inthe loading step, can include the steps of loading the column with afluid including the target compound, washing the column to removeunwanted material, eluting the target compound to the column, andre-equilibrating the column. In other examples, a cycle ofchromatography using a cationic and/or anionic exchange chromatographycolumn, where unwanted material binds to the chromatography resin duringthe loading step, while the target compound does not, can include thesteps of loading the column with a fluid including the target compound,collecting the compound in the flow-through, and re-equilibrating thecolumn. As is well-known in the art, any of the single steps in achromatography cycle can include a single buffer or multiple buffers(e.g., two or more buffers), and one or more of any of the single stepsin a chromatography cycle can include a buffer gradient. Any of thecombination of various well-known aspects of a single cycle ofchromatography can be used in these methods in any combination, e.g.,different chromatography resin(s), flow-rate(s), buffer(s), voidvolume(s) of the column, bed volume(s) of the column, volume(s) ofbuffer used in each step, volume(s) of the fluid including the targetprotein, and the number and types of buffer(s) used in each step.

The columns used for the column chromatography and elution can be anysuitable vessel of any cross-sectional shape that allows loading fromone end (e.g., top) and outflow from the other end (e.g., bottom).Typically, the column is tubular, having a cylindrical, nearcylindrical, or ellipsoidal shape. (e.g., a cylinder, near cylindricalshape, or ellipsoidal shape).

Optionally, the castanospermine preparation method further compriseschemically modifying the resulting castanospermine to make a derivativeof castanospermine.

Optionally, as with other active pharmaceutical ingredients, thecastanospermine or castanospermine derivative may be formulated as apharmaceutically acceptable salt to achieve desirable formulationproperties (Gupta D et al., “Salts of Therapeutic Agents: Chemical,Physiochemical, and Biological Considerations,” Molecules, 2018,23:1719; Makary, P., “Principles of Salt Formation”, UK Journal ofPharmaceutical and Biosciences, 2014, 2(4):01-04).

EXEMPLIFIED EMBODIMENTS Embodiment 1

A method for preparing castanospermine, comprising:

(a) separating a liquid fraction comprising castanospermine fromcastanospermine-containing solid plant material;

(b) purifying the castanospermine in the liquid fraction from (a) by:(i) mixing the liquid fraction with an ion exchange medium (e.g., ionexchange resin or ion exchange polymer) under conditions that allowcastanospermine in the liquid fraction to be bound to the resin, or (ii)column chromatography using a chromatography medium;

(c) eluting the castanospermine from the ion exchange medium of (b)(i)or chromatography medium of (b)(ii) to produce an eluate; and

(d) purifying the castanospermine in the eluate of (c) by mixing theeluate with an ion exchange medium (e.g., ion exchange resin or ionexchange polymer) under conditions that allow castanospermine in theeluate to be bound to the ion exchange medium, and eluting thecastanospermine.

Embodiment 2

The method of embodiment 1, wherein the castanospermine-containing plantmaterial comprises castanospermine-containing plant seed material, andwherein the castanospermine-containing plant seed material is preparedfor said separating by grinding seeds lacking seed cases, producingground particles of a size no larger than about 2 millimeters indiameter, and preferably homogenized.

Embodiment 3

The method of embodiment 1, wherein the castanospermine-containing solidplant material is any castanospermine-containing part or parts ofCastanospermum australe, Alexa spp., or a combination thereof.

Embodiment 4

The method of embodiment 1, wherein the castanospermine-containing solidplant material comprises or consists of solid Castanospermum australeseed material.

Embodiment 5

The method of embodiment 4, wherein C. australe seed material isprepared by grinding C. australe seeds lacking seed cases, producingground particles of a size no larger than about 2 millimeters indiameter.

Embodiment 6

The method of any preceding embodiment, wherein said separating of step(a) includes an ethanol extraction; or wherein said separating of step(a) includes a heat treatment comprising heating thecastanospermine-containing solid plant material (e.g., seeds)sufficiently to denature starch within the plant material (e.g., about63 to 77 degrees C.), allowing the heat-treated plant material to cool,grinding and/or cutting the cooled plant material, and carrying outwater extraction on the plant material to obtain the liquid fractioncomprising castanospermine.

Embodiment 7

The method of embodiment 1, wherein said separating of step (a)comprises: (i) allowing the solid material to settle out and placing(e.g., pumping) most of the liquid (first recovered fraction) into acontainer (e.g., a vat or other vessel) followed by filtration orcentrifugation of the remaining material to remove the rest of theliquid fraction (second recovered fraction) and combining the secondrecovered fraction with the first recovered fraction; or (ii) directlyseparating the liquid fraction from the solid material using acontinuous process such as centrifugation or filtration.

Embodiment 8

The method of any preceding embodiment, wherein the separating of step(a) is repeated one or more times, pooling the obtained liquid fractionsfrom each separation.

Embodiment 9

The method of any preceding embodiment, wherein the ion exchange mediumof step (b) is an acidic cation exchange medium (e.g., DOWEX-50 H⁺).

Embodiment 10

The method of any preceding embodiment, wherein the ion exchange mediumof step (b) comprises ion exchange beads.

Embodiment 11

The method of any preceding embodiment, further comprising sampling themixture after mixing for a duration of time in (b)(i), and measuring theamount of castanospermine remaining in the liquid phase (such as byliquid chromatography mass spectrometry (LC/MS)).

Embodiment 12

The method of any preceding embodiment, further comprising assaying theamount of castanospermine in the eluted fractions (e.g., by LC/MS) insaid eluting step (c) and pooling those fractions with desired highamounts of castanospermine present in order to minimize the amount ofimpurities carried to the next step.

Embodiment 13

The method of any preceding embodiment, wherein the solvent used forsaid eluting step of (c) is ammonium hydroxide (NH₄OH).

Embodiment 14

The method of any preceding embodiment, further comprising removingresidual solvent after said eluting of step (c) and before saidpurifying of (d).

Embodiment 15

The method of any preceding embodiment, wherein said purifying step of(d) is carried out batch-wise, or over a column or other vessel.

Embodiment 16

The method of embodiment 1, wherein the ion exchange medium of saidpurifying step of (d) comprises a pyridinium ion form resin, whereincastanospermine is exchanged with the pyridinium ion, resulting incastanospermine bound to the resin and pyridine released into solution.

Embodiment 17

The method of any preceding embodiment, wherein castanospermine iseluted in said purifying step (d) using an elution solvent comprising:pyridine in deionized water, or ammonium hydroxide (NH₄OH).

Embodiment 18

The method of any preceding embodiment, wherein the purity ofcastanospermine after said purifying step of (d) is at least about 95%if the castanospermine is eluted by a column or other vessel, or about90% to about 95% if the castanospermine is eluted batch-wise.

Embodiment 19

The method of any preceding embodiment, comprising further purifyingeluate of step (d).

Embodiment 20

The method of embodiment 19, wherein said further purifying comprises:(i) repeating the purifying step of (d) on the eluate, or (ii)recrystallizing the castanospermine enriched crystals in the eluate ofstep (d) with a solvent, such as ethanol, optionally sonicating, placingthe solution at reduced temperature to stimulate crystal formation, forharvesting and drying.

Embodiment 21

The method of any preceding embodiment, further comprising regeneratingthe ion exchange medium of step (d).

Embodiment 22

The method of any preceding embodiment, further comprising measuring theamount of castanospermine in the product of one or more of steps (a),(b), (c), or (d).

Embodiment 23

The method of embodiment 22, wherein said measuring is conducted byliquid chromatography mass spectrometry (LC/MS).

Definitions

The term “a,” “an,” “the” and similar terms used in the context of thepresent invention (especially in the context of the claims) are to beconstrued to cover both the singular and plural unless otherwiseindicated herein or clearly contradicted by the context. For example,the term “compound” includes a singular compound and a plurality ofcompound unless specified to the contrary. Furthermore, to the extentthat the terms “including”, “includes”, “having”, “has”, “with”, orvariants thereof are used in either the detailed description and/or theclaims, such terms are intended to be inclusive in a manner similar tothe term “comprising”. The transitional terms/phrases (and anygrammatical variations thereof) “comprising”, “comprises”, “comprise”,“consisting essentially of”, “consists essentially of”, “consisting” and“consists” can be used interchangeably.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid,hydrofluoric acid, phosphorous acid, and the like. Also included aresalts that are formed with organic acids such as aliphatic mono- anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, alkanedioic acids, aromatic acids, aliphatic and. aromaticsulfonic acids, etc. and include, for example, acetic acid,trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like. Exemplary salts thus include sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates,trifluoroacetates, propionates, caprylates, isobutyrates, oxalates,malonates, succinate suberates, sebacates, fumarates, maleates,mandelates, benzoates, chlorobenzoates, methylbenzoates,dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates,phenylacetates, citrates, lactates, malates, tartrates,methanesulfonates, and the like. Also contemplated are salts of aminoacids, such as arginates, gluconates, and galacturonates (see, forexample, Berge S. M. et al., “Pharmaceutical Salts,” Journal ofPharmaceutical Science, 66:1-19 (1997), which is hereby incorporated byreference in its entirety). Acid addition salts of basic compounds maybe prepared by contacting the free base forms with a sufficient amountof the desired acid to produce the salt according to methods andtechniques with which a skilled artisan is familiar.

“Pharmaceutically acceptable base addition salt” refers to those saltsthat retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Pharmaceutically acceptable base addition salts may beformed with metals or amines, such as alkali and alkaline earth metalsor organic amines. Salts derived from inorganic bases include, but arenot limited to, sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Salts derived from organic bases include, but are not limited to, saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, for example, isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, ethanolamine,diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline,betaine, ethylenediamine, ethylenedianiline, N-methylglucamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like. See Bergeet al., supra.

As used herein, a “derivative” or “pharmaceutically active derivative”refers to any compound that upon administration to the recipient, iscapable of providing directly or indirectly, the activity ofcastanospermine (e.g., anti-viral activity, alpha-glucosidase inhibitoryactivity, such as inhibition of alpha-glucosidase I). The term“indirectly” also encompasses “prodrugs” which may be converted to theactive form of the drug, e.g., via endogenous enzymes or metabolism(biotransformation). The prodrug is a derivative of the castanospermineand presenting desired activity (e.g., anti-viral activity,alpha-glucosidase inhibitory activity) that has a chemically ormetabolically decomposable group, and a compound that may be convertedinto a pharmaceutically active compound according to the invention invivo by solvolysis under physiological conditions. The prodrug isconverted into castanospermine by a reaction with an enzyme, gastricacid or the like under a physiological condition in the living body,e.g., by oxidation, reduction, hydrolysis or the like, each of which iscarried out enzymatically. These compounds can be produced fromcastanospermine according to well-known methods. The term “indirectly”also encompasses metabolites of compounds according to the invention.Chemical reactions, reactants, and reagents useful for makingderivatives can be found, for example, in March's Advanced OrganicChemistry, 7^(th) edition, 2013, Michael B. Smith, which is incorporatedherein by reference in its entirety.

More specifically, the term “prodrug” refers to a chemical compound thatcan be converted by the body (i.e., biotransformed) to another chemicalcompound that has pharmacological activity. The prodrug may itself havepharmacological activity before conversion, or be inactive beforeconversion and activated upon conversion. Active prodrugs or inactiveprodrugs of compounds of the invention may be administered to a subjector contacted with a cell in vitro or in vivo. Instead of administering adrug directly, a prodrug may be used instead to improve how a drug isabsorbed, distributed, metabolized, and excreted (ADME). For example, aprodrug may be used to improve bioavailability when a drug itself ispoorly absorbed from the gastrointestinal tract, or to improve howselectively the drug interacts with cells or processes that are not itsintended target, which can reduce adverse or unintended effects of adrug. Major types of prodrugs include, but are not limited to, type Iprodrugs, which are biotransformed inside cells (intracellularly), andtype II prodrugs, which are biotransformed outside cells(extracellularly), such as in digestive fluids or in the body'scirculatory system. These types can be further categorized into subtypesbased on factors such as whether the intracellular bioactivationlocation is also a site of therapeutic action, or whether or notbioactivation occurs in the gastrointestinal fluids or in thecirculation system (Wu, Kuei-Meng, “A New Classification of Prodrugs:Regulatory Perspectives, Pharmaceuticals, 2009, 2(3):77-81, which isincorporated by reference herein in its entirety).

The term “metabolite” refers to all molecules derived fromcastanospermine in a cell or organism, preferably mammal.Pharmaceutically active metabolites of the compounds of the inventionmay be administered to a subject or contacted with a cell in vitro or invivo.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

Pharmaceutical formulations include “pharmaceutically acceptable” and“physiologically acceptable” carriers, diluents or excipients. In thiscontext, the terms “pharmaceutically acceptable” and “physiologicallyacceptable” include solvents (aqueous or non-aqueous), solutions,emulsions, dispersion media, coatings, isotonic and absorption promotingor delaying agents, compatible with pharmaceutical administration. Suchformulations can be contained in a liquid; emulsion, suspension, syrupor elixir, or solid form; tablet (coated or uncoated), capsule (hard orsoft), powder, granule, crystal, or microbead. Supplementary compounds(e.g., preservatives, antibacterial, antiviral and antifungal agents)can also be incorporated into the compositions.

The phrase “effective amount” means an amount of an agent, such ascastanospermine or a derivative thereof, that (i) treats or prevents theparticular disease, condition, or disorder, (ii) attenuates,ameliorates, or eliminates one or more symptoms of the particulardisease, condition, or disorder, (iii) prevents or delays the onset ofone or more symptoms of the particular disease, condition, or disorderdescribed herein. Castanospermine has been shown to inhibit infection bycertain viruses in vitro and/or in vivo (Whitby K et al., J Virol., July2005, 79(14):8698-8706; U.S. Pat. No. 10,561,642). Thus, in the contextof castanospermine or a derivative thereof, an “effective amount” mayalso mean an amount that (iv) inhibits viral infection in vitro and/orin vivo (e.g., flavivirus infection such as Dengue virus), or (v)inhibits one or more types of glucosidases in vitro or in vivo.

Castanospermine, derivatives, and pharmaceutically acceptable salts ofcastanospermine and derivatives produced using the method of theinvention, and compositions comprising them, can be formulated accordingto known methods for preparing pharmaceutically useful compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.

In general, the compositions can be formulated such that an effectiveamount of the castanospermine or derivative thereof is combined with asuitable carrier in order to facilitate effective administration of thecomposition. The compositions used can also be in a variety of forms.These include, for example, solid, semi-solid, and liquid dosage forms,such as tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also may include conventionalpharmaceutically acceptable carriers and diluents which are known tothose skilled in the art. Examples of carriers or diluents for use withthe castanospermine and its derivatives include, but are not limited to,water, saline, oils including mineral oil, ethanol, dimethyl sulfoxide,gelatin, cyclodextrans, magnesium stearate, dextrose, cellulose, sugars,calcium carbonate, glycerol, alumina, starch, and equivalent carriersand diluents, or mixtures of any of these. Formulations ofcastanospermine and derivatives thereof can also comprise suspensionagents, protectants, lubricants, buffers, preservatives, andstabilizers. To provide for the administration of such dosages fortherapeutic treatment, pharmaceutical compositions of the invention maycomprise between about 0.1% and 45%, and especially, 1 and 15% by weightof the total of the castanospermine, and optionally other activeingredients, based on the weight of the total composition includingcarrier or diluent.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following is an example that illustrates procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example—Large-Scale Purification of Castanospermine from Castanospermumaustrale Seeds

This method can be adjusted to accommodate any needed scale of theproduction process.

1) Grinding Seeds.

Seed cases of the seeds of the Castanospermum australe tree are removedand discarded, leaving just the seed (generally oval shaped at about 3-5cm in diameter and 2 cm thick). The seeds are ground using any methodsuitable for the scale of purification being conducted. Multiple passesthrough the grinder or grinding process can be used to generate a finemoist meal. The smaller the size of the ground particles the better andshould be no larger than about 2 mm in size.

2) Homogenizing Ground Seed Meal.

Ground seed meal is mixed with 1-2 volumes (v/v) of 75% ethanol(prepared by mixing 95% EtOH with deionized water and adjusting the EtOHconcentration to 75% by use of a hydrometer) and vigorously homogenizedfor 10-12 minutes. Other ethanol concentrations may be used forextraction, provided the final ethanol concentration is sufficient toprecipitate the starch from the plant material. Any suitablehomogenization method can be used based upon the scale being processed.The intent is to thoroughly extract the seed meal and reduce the size ofthe particles to roughly the size of ground coffee.

Once the homogenization step is completed the liquid is removed from thesolid material. This can be accomplished in a number of ways to include:a) allowing the solid material to settle out and pump most of the liquidinto a large vat or vessel followed by filtration by any suitable meansor centrifugation of the remaining material to remove the rest of theliquid fraction and is combined the liquid recovered earlier; b)separating the liquid fraction from the solid material directly by acontinuous process such as centrifugation or filtration; or c) any othermeans of separating the liquid fraction from the solid material suitablefor use in the scale of the process being used.

After completion of the first extraction step, the recovered solidmaterial will be a moist cake. This can optionally be extracted a secondtime using an additional 2-3 volumes (v/v) of 75% ethanol and theextraction process repeated. In the end, all liquid fractions can bepooled and processed in the next step (purification). The twiceextracted solid material can be discarded. While a third extraction willlikely yield additional castanospermine; it is not cost-effective.

Optional Heat Treatment as Alternative to 1) and 2), Above:

This optional heat treatment has the advantage of not using ethanol forextraction from the seeds, and would just be utilized for the extractionphase.

The procedure involves heating raw Castanospermum seeds to an internaltemperature sufficient to denature starch and other components (e.g.,about 63 to about 77 degrees C.) and allowing them to cool. This heattreatment will render the starch and other components mostly insolublein water (by contrast, ethanol in extraction fluid is largely there toprecipitate starch and other components so they can be filtered awaymore easily). Castanospermine is stable to this heat treatment.

The cooled seeds are then ground or cut up to yield pieces about 2 to 4mm in diameter and placed in deionized water. This mixture can either bestirred to keep the seed pieces suspended or more aggressively mixed tobreak the pieces up, for example, in a blender. The more gentle stirringwill require more extraction time for the castanospermine to leach outinto the water, for example, overnight but will leave solid residue thatwill settle out on its own and not require filtration or centrifugationto obtain a clear or slightly turbid aqueous solution containingextracted castanospermine. More aggressive mixing or blending will notrequire an extensive extraction time for the castanospermine to becomedissolved in water but, after allowing solid residue to settle, theaqueous solution may have considerable particulate material to filter orcentrifuge out. The solid residue can optionally be re-extracted withadditional deionized water.

From either option the pooled aqueous solution can then be mixed with anion exchange resin such as Dowex 50 H+ form, either batch wise withstirring or poured into a column, as described in 3), below. Aftermixing with the resin or passing through the resin, the aqueous solutionis discarded and the resin extensively washed with deionized water andeluted with an elution solvent (e.g., 2N NH₄OH) as described elsewhere.Subsequent steps are the same as if ethanol had been used for theinitial extraction.

3) Treatment with Dowex-50 H⁺ Form.

Batchwise Treatment:

Depending on the separation method used to obtain the liquid fractionthere may be very fine particles or “flour” remaining in the liquidfraction. This will settle out and can easily be removed from the clearliquid prior to the next step. However, it is not critical or necessaryto remove the fine particles as they will be eliminated in a later step.

The liquid is mixed with Dowex-50 H⁺ form ion exchange resin (or othersimilar strongly acidic cation exchange resin), 25-50 mesh or othersuitable size of resin (very small resin particles, 100 mesh or smallerwill be inconvenient in subsequent steps and lead to losses of resin andcastanospermine) in approximate proportions of 1 liter of packed resinplus the liquid derived from the extraction of 3.5 Kg of ground seedmeal. This is then placed in a suitably sized container and stirredunder adequate conditions which allow the resin to remain suspended inthe liquid and not settle to the bottom. This proportion of resin toliquid extract necessary will vary depending on the castanosperminecontent of the seeds being extracted. Generally the castanosperminecontent of the raw seeds is about 0.5% to 1% by weight. Accordingly, theamount of resin and liquid extract appropriate for a given batch ofseeds can be optimized by sampling the liquid after stirring for ½ to 1hour and testing the amount of castanospermine remaining in the liquidphase by LC/MS (see under “LC/MS assay” below).

Based on this, an optimal amount of liquid extract to resin ratio can bedetermined that is capable of yielding >90-95% of the castanosperminebound to the resin and the period of time the mixture must be stirred toachieve that result. This proportion should be relevant for anysubsequent extractions with a given batch of seeds. This is helpful tobegin reducing the scale of the process in subsequent steps.

After the castanospermine has been absorbed on to the resin, the resincan be allowed to settle to the bottom of the vessel being used and theliquid fraction poured or pumped off. This liquid, generally about 60%EtOH, can be recycled by distillation to obtain clear, colorless EtOHand used again after adjusting the EtOH concentration with deionizedwater to 75% through the use of a hydrometer.

The resin, which may be turbid from the seed residue and “flour”, iswashed extensively with deionized water until the liquid becomes clearand lacks turbidity. Washing is done by thorough stirring of the resinand suspending it in deionized water, allowing the resin to settle, andremoving the water by any suitable method depending on scale. It isimportant to do this quickly to minimize the amount of seed residue thatis able to settle with the resin before removal of the water wash. Oncethe resin is thoroughly washed and no turbidity remains in the waterwash, the resin can be optionally transferred to a vessel capable ofdraining from the bottom and being able to be filled from the top. Itcan be an open vessel or sealed vessel that allows fluid to flow throughthe resin bed either by gravity or by pumping. The size and shape of thevessel to be used is based upon the volume needs for the amount of resinbeing used. A column containing either straight or tapered sides isideal. The bottom of the column or other vessel must have a porousbarrier that will not allow the resin to pass through while allowing theeluting liquid to freely flow. To monitor progress of the elution stepit is convenient to affix some heat sensitive tape (for example whichchanges color when warmed) to the side of the vessel containing theresin.

Column Treatment:

An alternate method for initial purification of 75% ethanol extractedseed meal is via column chromatography depending on scale, availableequipment, and individual preferences.

To apply column chromatography the biggest difference from the batchwisemethod is that all of the particulate and insoluble “flour” from theseed should be completely removed prior to loading the column. Thismaterial can otherwise clog the column and severely reduce or stop theflow rate. Removal can be done in a variety of ways or steps. Theextract can be allowed to stand for an adequate period of time for thisinsoluble material to settle to the bottom of the container. And/or, theextract can be either filtered or centrifuged, for example in acontinuous flow centrifuge, to yield clarified extract capable of beingloaded onto the resin which is either in a column or other suitablevessel which allows loading from one end (e.g. top) and outflow from theother end (e.g. bottom). The size of the column and amount of resin touse is based on the scale of the preparation and can be adjusted asdescribed above to optimize the amount of seed meal that can beprocessed at a time. The column is loaded and washed extensively withdeionized water until the effluent is clear and the conductivity of themixture is similar to deionized water indicating any unboundcontaminants have been eliminated. The column is then eluted and thecastanospermine is further processed as described below.

4) Elution of Castanospermine from Dowex 50 H⁺ Form.

After transferring the batch-washed resin to an elution vessel (asnecessary), the resin is further washed with water to remove anyadditional turbidity that may remain. It is then eluted using 2M NH₄OH.Depending on the size and dimensions of the elution vessel being used,it is often desirable to place a porous barrier such as filter paper orthe like on the top of the resin bed to minimize disruption of the resinbed as the elution solvent is added to the vessel. As the NH₄OH goesdown the column the heat of neutralization will create warming allowingthe progress of the elution to easily be monitored. After the warmingfront reaches about half way down the vessel, fractions of any practicalsize can be collected. The castanospermine elutes from the column in theregion of the warming front and fractions just prior to it flowing fromthe column. This can be conveniently monitored by heat sensitive tapeaffixed to the side of the elution vessel. Once the warming front haspassed through the column an odor of ammonia will be present and all ofthe castanospermine is off the column. Additional NH₄OH can be passedover the resin to elute general impurities. It is possible to assay theamount of castanospermine in the eluted fractions by LC/MS and pool justthose fractions with high amounts of castanospermine present to minimizethe amount of impurities carried along to the next step.

The resin, after eluting with a desired amount of additional 2 M NH₄OHis then washed extensively with deionized water followed by treatmentwith 2 N HCl or 2 M H₂SO₄ to convert the resin back to the H⁺ form.Adequate acid is put over the resin until the pH eluting from the bottomis strongly acidic. At that point the resin is washed extensively withwater until the pH is no longer strongly acidic (it may go to pH 5 orso).

It is possible to alternately do all elution steps of the Dowex 50 H⁺form resin using 2N NH₄OH in a batchwise fashion and separating thecastanospermine containing supernatant fraction from the resin using aconvenient method.

Once the Dowex 50 resin has been converted back to the H⁺ form andextensively water washed (for a detailed procedure see below under“Regeneration of Dowex 50 resin”), it can be used again (and again) forabsorption of castanospermine from additional liquid ethanol extract ofseed meal following the same procedures described above.

5) Preparation of the Castanospermine Containing Dowex 50 H⁺ Form ElutedFractions for Subsequent Steps.

The fractions from the above step which contain the desired levels ofcastanospermine (which can be determined using LC/MS analysis) arepooled. It is possible that some NH₄OH may be present which couldinterfere with efficient castanospermine binding in the nextpurification step. Residual NH₄OH can be removed by any means capable ofevaporating the residual NH₄OH. One method is via concentration using arotary evaporator. It is only necessary to treat (or concentrate) thepooled fractions until all odor from NH₄OH has been removed. Duringrotary evaporation (or any other method of concentration or removal ofNH₄OH) it is possible to heat the pooled fractions to at least as muchas 80° C. without harming (degrading) the castanospermine.

6) Purification on Dowex 50 Pyridinium Ion Form Resin.

The castanospermine containing concentrate from the Dowex 50 H⁺ resinelution is then mixed (either batchwise or over a column or other vesselcontaining a means for liquids to flow from the top and outflow throughthe bottom) with Dowex 50 resin in the pyridinium ion form (again, 25-50mesh is suitable). This resin forms a separate batch of Dowex 50 resinused only for this purpose and is kept separate from the resin used inthe first step. Varying amounts of resin can be used based upon thescale of the purification being conducted. An amount of resin to use canbe estimated based upon the total exchange capacity of the resin andestimates of the amount of castanospermine present in the elutedfractions of the previous step. This amount can be roughly determined byLC/MS assays of castanospermine. The resin can be prepared by passingpyridine in deionized water (for example, 2 M pyridine) over Dowex 50 H⁺form resin in a column or similar vessel, or by adding it to the resinbatchwise. Adequate pyridine is passed over the resin in a column or ismixed with the resin batchwise until an odor of pyridine is apparent inthe column outflow or in the liquid portion of the batchwise treatment.The resin is then washed extensively with deionized water until theresidual pyridine odor is not apparent or is very low. The resin canthen be used in the next step.

Pooled fractions that eluted with 2N NH₄OH from the initial Dowex 50 H⁺form resin (conducted either batchwise or using a column or vesselcapable of allowing elution solvent to flow through the resin and outthe bottom), after removal of excess NH₄OH, are contacted with deionizedwater washed Dowex 50 pyridinium ion form resin (for example, 25-50mesh) either batchwise or by use of a column or similar vessel allowingsolvent flow through the resin and out the bottom. This step exchangescastanospermine with the pyridinium ion resulting in pyridine releaseinto solution of the pass-through or batch treatment supernatant. Ifdesired, the amount of castanospermine present in the columnpass-through or in the batch treatment supernatant can be determined byLC/MS assay to ensure the resin is not overloaded with castanospermineor to ensure that the maximum amount of castanospermine has been loadedonto the resin for efficient processing.

Excess pyridine can be recovered from any suitable resin wash, columnpass-through or batchwise treatment supernatant, or other suitablepyridine containing solution by distillation under reduced pressure suchas in a rotary evaporator. Pyridine forms a minimum boiling azeotropewith water allowing simple recovery of excess pyridine. Recoveredpyridine can be used to convert Dowex 50 H⁺ resin to the pyridinium formas needed.

Once this resin has been loaded with castanospermine as desired andwashed extensively with deionized water so that the wash is clear andcolorless there are two potential methods for elution. The firstinvolves elution (either batchwise or via running solvent through acolumn) using 2M pyridine in deionized water. In large scalepurification procedures castanospermine slowly elutes from the resinover 2 to 3 resin bed volumes of solvent (or more if done batchwise)requiring a significant quantity of pyridine and creating a significantvolume of pyridine containing fluid for pyridine recovery. Fractionscontaining castanospermine can be identified and quantitated by LC/MS orby concentrating a portion to dryness using a rotary evaporator toreveal a white or yellowish precipitate in the evaporator flask. Adecision can be made on when to stop the elution based on these results.Castanospermine containing fractions are pooled and concentrated to neardryness, for example, using a rotary evaporator and the castanospermineis then crystallized using either absolute or 95% ethanol and placingthe mixture at low temperature (for example, in a freezer) to stimulatecrystal formation. Sonication of the crystallization vessel, for examplewith a bath sonicator, can be used to stimulate crystal formation. Theresulting crystals can be harvested by filtration, dried, and storedmost suitably in the dark in a freezer. Typical purity of the isolatedcastanospermine at this step is about 95% or greater.

A second method for elution of the Dowex 50 pyridinium ion form columnis with 2 N NH₄OH. Under this condition the castanospermine elutesrapidly and with a lower volume of elution solvent. If using a column orsimilar vessel, collection of fractions should be started immediately.Fraction sizes can be set at any volume convenient for the scale of theprocess being used. Progress of the elution can be monitored byfollowing the heating front from NH₄OH binding to the resin down thecolumn. Once the heating front has come off the column an odor of NH₄OHwill be apparent and collection of fractions should be stopped.Additional NH₄OH can be run over the column to wash additional coloredcontaminants off the column, and discarded. The castanosperminecontaining fractions can be identified as described above, pooled,reduced in volume to near dryness, crystalized using absolute or 95%ethanol, and harvested and dried as described above. If using a methodfor reducing the volume of castanospermine containing fractions thatinvolves warming the solution, for example, under reduced pressure in arotary evaporator, castanospermine remains stable in temperatures ofabout 80° C. to 90° C. Alternatively, batch elution with 2N NH₄OH can beconducted using a suitable number of batch elution volumes needed torecover the eluting castanospermine. One consequence of batch elutionwith 2N NH₄OH compared to use of a column is that the purity can bereduced. Column eluted castanospermine should be about 95% pure whereasbatch eluted castanospermine might be 90% to 95% pure at this stage.

7) Further Purification of Castanospermine.

Depending on the purity needs for the isolated castanospermine,additional purification efforts may be needed. Two general methodsshould be tried initially, recrystallization in ethanol or a repeat ofthe Dowex 50 pyridinium ion form column step. For recrystallization,crude castanospermine enriched crystals are dissolved in a minimumvolume of water and 5- to 10 volumes of absolute or 95% ethanol isadded, optionally sonicated, and placed at low temperature (for examplein a freezer) to stimulate crystal formation. Crystals are harvested anddried as described above.

Use of a second Dowex 50 pyridinium ion form step is best conductedusing a separation column or similar device allowing flow through ofsolvent through the resin. A separate batch of Dowex 50 pyridinium ionform resin than used in the first such step should be used. This batchof resin can be repeatedly used for this purpose after regeneration asdescribed elsewhere. Crude castanospermine crystals are dissolved indeionized water and loaded onto the column. The capacity of the resinwill dictate the maximum amount that can be treated at one time. Thiscan be determined as described above. The column is loaded and washedextensively with deionized water until the effluent is colorless. It isthen eluted as before with 2N NH₄OH. Castanospermine containingfractions are determined, pooled, concentrated, and the castanosperminecrystallized and harvested as described above. Such re-purifiedcastanospermine should be mostly white or very lightly yellow coloredand in ≥95% purity at this stage. If castanospermine was eluted using 2Mpyridine in the first Dowex 50 pyridinium ion form resin purificationstep and processed again on a second Dowex 50 pyridinium ion form resinstep using elution with 2N NH₄OH, white crystals should be obtained thatare ≥98% pure castanospermine. After crystallization, harvesting, anddrying of the crystals they should be stored in the dark and preferablyin a freezer.

8) Regeneration of Dowex 50 Resin Used in Castanospermine Purification.

Dowex 50 resin is used in several purification steps and it can beregenerated time and again for repeated use. If purity of the finalproduct trends down it might be because of the presence and buildup ofadsorbed impurities on the resin after repeated use which could justifydiscarding that resin and starting afresh.

The first step in regeneration of eluted resin is to wash it extensivelywith water to remove all prior elution solvent and any colored materialspresent. The resin is then treated with either 2N HCl or 2M H2SO4 indeionized water until the pH of the effluent or batch treatment solutionbecomes strongly acidic. This is then followed by an additionalextensive deionized water wash until the pH rises to about pH5 or higherindicating all free acid has been washed away. For resin intended forthe first purification step, this resin is now ready to be used.

To convert resin to the pyridinium ion form, the recycled Dowex 50 H⁺form resin just prepared is treated with 2N pyridine (the exactconcentration is not critical and either fresh or recycled pyridine orsome of both can be used). The resin is treated with pyridine until apyridine odor can be detected in the column effluent or batch treatmentsupernatant depending on the method used. Then, the resin is washedextensively with deionized water until little or no pyridine odor isdetected. It is then ready for use in the appropriate purificationsteps.

9) LC/MS Assay for Castanospermine.

Typical conditions for LC/MS analysis of castanospermine are as follows.

Sample: a solution of Castanospermine (MW: 189.2) in H₂O

MS: Agilent 6410 triple quadrupole mass spectrometer/AP ESI scan mode,or similar

HPLC: Agilent HP1200 with Thermo betasil phenyl column, or similar,under isocratic condition with mobile phase solvent of 0.1% formic acidin 5% Acetonitrile and 95% H2O, column temp: 50 C, flow rate: 0.25mL/min, 5 uL injection.

FIG. 1 : chromatogram

FIG. 2 : mass spectrum (MH+: 190.1, MH−H2O+: 172.2, MH−2H2O+: 154.2,MH−3H2O+: 136.1, M+Na+: 212.1)

Purity of castanospermine can be assessed based upon the chromatogram,and quantity can be assessed based upon the size of the MH+ ion at 190.1AMU in comparison with dilutions and a standard curve.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

We claim:
 1. A method for preparing castanospermine, comprising: (a)separating a liquid fraction comprising castanospermine fromcastanospermine-containing solid plant material, wherein thecastanospermine-containing solid plant material is anycastanospermine-containing part or parts of Castanospermum australe,Alexa spp., or a combination thereof; (b) purifying the castanosperminein the liquid fraction from (a) by: (i) mixing the liquid fraction withan ion exchange medium (e.g., ion exchange resin or ion exchangepolymer) under conditions that allow castanospermine in the liquidfraction to be bound to the resin, or (ii) column chromatography using achromatography medium; (c) eluting the castanospermine from the ionexchange medium of (b)(i) or chromatography medium of (b)(ii) to producean eluate; and (d) purifying the castanospermine in the eluate of (c) bymixing the eluate with an ion exchange medium (e.g., ion exchange resinor ion exchange polymer) under conditions that allow castanospermine inthe eluate to be bound to the ion exchange medium, and eluting thecastanospermine.
 2. The method of claim 1, wherein thecastanospermine-containing plant material comprisescastanospermine-containing plant seed material, and wherein thecastanospermine-containing plant seed material is prepared for saidseparating by grinding seeds lacking seed cases, producing groundparticles of a size no larger than about 2 millimeters in diameter, andpreferably homogenized.
 3. The method of claim 1, wherein thecastanospermine-containing solid plant material is anycastanospermine-containing part or parts of Castanospermum australe,Alexa spp., or a combination thereof.
 4. The method of claim 1, whereinthe castanospermine-containing solid plant material comprises orconsists of solid Castanospermum australe seed material.
 5. The methodof claim 4, wherein C. australe seed material is prepared by grinding C.australe seeds lacking seed cases, producing ground particles of a sizeno larger than about 2 millimeters in diameter.
 6. The method of claim1, wherein said separating of step (a) includes an ethanol extraction;or wherein said separating of step (a) includes a heat treatmentcomprising heating the castanospermine-containing solid plant materialsufficiently to denature starch within the plant material, allowing theheat-treated plant material to cool, grinding and/or cutting the cooledplant material, and carrying out water extraction on the plant materialto obtain the liquid fraction comprising castanospermine.
 7. The methodof claim 1, wherein said separating of step (a) comprises: (i) allowingthe solid material to settle out and placing most of the liquid (firstrecovered fraction) into a container followed by filtration orcentrifugation of the remaining material to remove the rest of theliquid fraction (second recovered fraction) and combining the secondrecovered fraction with the first recovered fraction; or (ii) directlyseparating the liquid fraction from the solid material using acontinuous process such as centrifugation or filtration.
 8. The methodof claim 1, wherein the separating of step (a) is repeated one or moretimes, pooling the obtained liquid fractions from each separation. 9.The method of claim 1, wherein the ion exchange medium of step (b) is anacidic cation exchange medium.
 10. The method of claim 1, wherein theion exchange medium of step (b) comprises ion exchange beads.
 11. Themethod of claim 1, further comprising sampling the mixture after mixingfor a duration of time in (b)(i), and measuring the amount ofcastanospermine remaining in the liquid phase.
 12. The method of claim1, further comprising assaying the amount of castanospermine in theeluted fractions in said eluting step (c) and pooling those fractionswith desired high amounts of castanospermine present in order tominimize the amount of impurities carried to the next step.
 13. Themethod of claim 1, wherein said purifying step of (d) is carried outbatch-wise, or over a column or other vessel.
 14. The method of claim 1,wherein the ion exchange medium of said purifying step of (d) comprisesa pyridinium ion form resin, wherein castanospermine is exchanged withthe pyridinium ion, resulting in castanospermine bound to the resin andpyridine released into solution.
 15. The method of claim 1, whereincastanospermine is eluted in said purifying step (d) using an elutionsolvent comprising: pyridine in deionized water, or ammonium hydroxide(NH₄OH).
 16. The method of claim 1, wherein the purity ofcastanospermine after said purifying step of (d) is at least about 95%if the castanospermine is eluted by a column or other vessel, or about90% to about 95% if the castanospermine is eluted batch-wise.
 17. Themethod of claim 1, comprising further purifying eluate of step (d). 18.The method of claim 17, wherein said further purifying comprises: (i)repeating the purifying step of (d) on the eluate, or (ii)recrystallizing the castanospermine enriched crystals in the eluate ofstep (d) with a solvent, such as ethanol, optionally sonicating, placingthe solution at reduced temperature to stimulate crystal formation, forharvesting and drying.
 19. The method of claim 1, further comprisingregenerating the ion exchange medium of step (d).
 20. The method ofclaim 1, further comprising measuring the amount of castanospermine inthe product of one or more of steps (a), (b), (c), or (d).